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Performance Evaluation of SHOX2 and RASSF1A Methylation for the Aid in Diagnosis of Lung Cancer Based on the Analysis of FFPE Specimen.

ABSTRACT: Emerging molecular diagnostic methods are more sensitive and objective, which can overcome the intrinsic failings of morphological diagnosis. Here, a RT-PCR-based in vitro diagnostic test kit (LungMe^®) was developed and characterized to simultaneously quantify the DNA methylation of SHOX2 and RASSF1A in FFPE tissue specimens. The clinical manifestations were evaluated in 251 FFPE samples with specificity and sensitivity of 90.4 and 89.8%, respectively. Furthermore, the quantitative analysis shows that the degree of SHOX2 methylation was correlated with the stages of lung cancer, but not in the case of RASSF1A. Our observation indicated that the DNA methylation of SHOX2 and RASSF1A may play different roles in cancer development. Comparison of the methylation levels of SHOX2 and RASSF1A between cancer and cancer-adjacent specimens (n = 30), showed they have "epigenetic field defect". As additional clinical validation, the hypermethylation of SHOX2 and RASSF1A was detected not only in surgical operative specimens, but also in histopathological negative puncture biopsies. SHOX2 and RASSF1A methylation detection can be used to increase sensitivity and NPV, which provide us with a more accurate method of differential diagnosis and are likely to be rapidly applied in clinical examinations.

SUBMITTER: Shi J

PROVIDER: S-EPMC7793934 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Performance Evaluation of SHOX2 and RASSF1A Methylation for the Aid in Diagnosis of Lung Cancer Based on the Analysis of FFPE Specimen.

Shi Juanhong J Chen Xue X Zhang Long L Fang Xia X Liu Yuting Y Zhu Xuyou X Zhang Haoyang H Fan Lichao L Gu Jun J Zhang Suxia S She Bin B Han Hongxiu H Yi Xianghua X

Frontiers in oncology 20201214

Emerging molecular diagnostic methods are more sensitive and objective, which can overcome the intrinsic failings of morphological diagnosis. Here, a RT-PCR-based in vitro diagnostic test kit (LungMe®) was developed and characterized to simultaneously quantify the DNA methylation of SHOX2 and RASSF1A in FFPE tissue specimens. The clinical manifestations were evaluated in 251 FFPE samples with specificity and sensitivity of 90.4 and 89.8%, respectively. Furthermore ...[more]

PMID: 33425721

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Project description:ObjectiveMethylation of the promoters of SHOX2 and RASSF1A are potentially informative biomarkers for the diagnosis of early lung adenocarcinoma (LUAD). Abnormal methylation of SHOX2 and RASSF1A promoters may promote the occurrence and facilitate the progression of LUAD.Materials and methodsWe selected 54 patients with early LUAD and 31 patients with benign lung nodules as a NJDT cohort and evaluated their DNA methylation and mRNA sequencing levels. The DNA methylation sequencing, mRNA sequencing, and clinical data for patients with LUAD were obtained from The Cancer Genome Atlas, and served as a TCGA cohort. We evaluated the diagnostic potential of a SHOX2 and RASSF1A combined promoter methylation assay for detection of early LUAD in the NJDT cohort. Then we explored the promoter methylation levels of SHOX2 and RASSF1A and their gene expression between normal and tumor samples at different stages in both cohorts. Pathways enriched between tumor and normal samples of methylation-positive patients in the NJDT cohort were analyzed.ResultsIn the NJDT cohort, the sensitivity of the combined promoter methylation assay on tumor samples was 74.07%, the sensitivity on paired tumor and paracancerous samples was 77.78%, and the specificities in both contexts were 100%. The combined promoter methylation-positive patients had clinicopathologic features including older age, larger tumors, deeper invasion, and higher Ki-67 expression. In both cohorts, SHOX2 expression increased and RASSF1A expression decreased in tumor samples. The promoter methylation level of SHOX2 and RASSF1A was significantly higher in tumor samples at stage I-II than that in normal samples. The promoter methylation levels of these two genes were both negative associated with their expression in early tumor samples. In the NJDT cohort, methylation-positive patients of both individual SHOX2 and RASSF1A assays exhibited upregulation of folate acid metabolism and nucleotide metabolism in tumor samples. The SHOX2 methylation-positive and RASSF1A methylation-positive patients showed the downregulation of pathways related to cell proliferation and apoptosis and pathways involved in DNA repair, cell growth and cell adhesion, respectively.ConclusionThe combined promoter methylation assay for SHOX2 and RASSF1A can be used for screening and diagnosis of early LUAD, with good sensitivity and specificity. The promoter methylation levels of SHOX2 and RASSF1A were associated with their abnormal mRNA expression, and affected DNA instability, cell proliferation, apoptosis and tumor microenvironment in patients with LUAD.

Project description:BackgroundEpigenetic changes during ageing and their relationship with cancer are under the focus of intense research. RASSF1A and NORE1A are novel genes acting in concert in the proapoptotic pathway of the RAS signalling. While NORE1A has not been previously investigated in the human liver, recent reports have suggested that RASSF1A is frequently epigenetically methylated not only in HCC but also in the cirrhotic liver.MethodsTo address whether epigenetic changes take place in connection to age and/or to the underlying disease, we investigated RASSF1A and NORE1A gene promoter methylation by conventional methylation specific PCR and Real-Time MSP in a series of hepatitic and non-hepatitic livers harboring regenerative/hyperplastic (cirrhosis/focal nodular hyperplasia), dysplastic (large regenerative, low and high grade dysplastic nodules) and neoplastic (hepatocellular adenoma and carcinoma) growths.ResultsIn the hepatitic liver (chronic hepatitic/cirrhosis, hepatocellular nodules and HCC) we found widespread RASSF1A gene promoter methylation with a methylation index that increased from regenerative conditions (cirrhosis) to hepatocellular nodules (p < 0.01) to HCC (p < 0.001). In the non-hepatitic liver a consistent pattern of gene methylation was also found in both lesional (focal nodular hyperplasia and hepatocellular adenoma) and non-lesional tissue. Specifically, hepatocellular adenomas (HA) showed a methylation index significantly higher than that detected in focal nodular hyperplasia (FNH) (p < 0.01) and in non-lesional tissue (p < 0.001). In non-lesional liver also the methylation index gradually increased by ageing (p = 0.002), suggesting a progressive spreading of methylated cells over time. As opposed to RASSF1A gene promoter methylation, NORE1A gene was never found epigenetically alterated in both hepatitic and non-hepatitic liver.ConclusionWe have shown that in non-lesional, regenerative and neoplastic liver the RASSF1A gene is increasingly methylated, that this condition takes place as an age-related phenomenon and that the early setting and spreading over time of an epigenetically methylated hepatocyte subpopulation, might be related to liver tumorigenesis.

Project description:The Liang Bua hominins from Flores, Indonesia, have been the subject of intense scrutiny and debate since their initial description and classification in 2004. These remains have been assigned to a new species, Homo floresiensis, with the partial skeleton LB1 as the type specimen. The Liang Bua hominins are notable for their short stature, small endocranial volume, and many features that appear phylogenetically primitive relative to modern humans, despite their late Pleistocene age. Recently, some workers suggested that the remains represent members of a small-bodied island population of modern Austro-Melanesian humans, with LB1 exhibiting clinical signs of Down syndrome. Many classic Down syndrome signs are soft tissue features that could not be assessed in skeletal remains. Moreover, a definitive diagnosis of Down syndrome can only be made by genetic analysis as the phenotypes associated with Down syndrome are variable. Most features that contribute to the Down syndrome phenotype are not restricted to Down syndrome but are seen in other chromosomal disorders and in the general population. Nevertheless, we re-evaluated the presence of those phenotypic features used to support this classification by comparing LB1 to samples of modern humans diagnosed with Down syndrome and euploid modern humans using comparative morphometric analyses. We present new data regarding neurocranial, brain, and symphyseal shape in Down syndrome, additional estimates of stature for LB1, and analyses of inter- and intralimb proportions. The presence of cranial sinuses is addressed using CT images of LB1. We found minimal congruence between the LB1 phenotype and clinical descriptions of Down syndrome. We present important differences between the phenotypes of LB1 and individuals with Down syndrome, and quantitative data that characterize LB1 as an outlier compared with Down syndrome and non-Down syndrome groups. Homo floresiensis remains a phenotypically unique, valid species with its roots in Plio-Pleistocene Homo taxa.

Project description:Background. Assessment of DNA methylation of specific genes is one approach to the diagnosis of cancer worldwide. Early stage detection is necessary to reduce the mortality rate of cancers, including those occurring in the stomach. For this purpose, tumor cells in circulating blood offer promising candidates for non-invasive diagnosis. Transcriptional inactivation of tumor suppressor genes, like PCDH10 and RASSF1A, by methylation is associated with progression of gastric cancer, and such methylation can therefore be utilized as a biomarker. Methods. The present research was conducted to evaluate DNA methylation in these two genes using blood samples of gastric cancer cases. Clinicopathological data were also analyzed and cumulative survival rates generated for comparison. Results. High frequencies of PCDH10 and RASSF1A methylations in the gastric cancer group were noted (94.1% and 83.2%, respectively, as compared to 2.97% and 5.45% in 202 matched controls). Most patients (53.4%) were in severe stage of the disease, with a median survival time of 8.4 months after diagnosis. Likewise, the patients with metastases, or RASSF1A and PCDH10 methylations, had median survival times of 7.3, 7.8, and 8.4 months, respectively. A Kaplan-Meier analysis showed that cumulative survival was significantly lower in those cases positive for methylation of RASSF1A than in their negative counterparts. Similarly, whereas almost 100% of patients positive for PCDH10 methylation had died after five years, none of the negative cases died over this period. Notably, the methylations of RASSF1A and PCDH10 were found to be higher in the late-stage patients and were also significantly correlated with metastasis and histology. Conclusions. PCDH10 and RASSF1A methylations in blood samples can serve as potential non-invasive diagnostic indicators in blood for gastric cancer. In addition to RASSF1A methylation, tumor stage proved to be a major prognostic factor in terms of survival rates.

Project description:ObjectivesSurgery with curative intent is the standard treatment for stage I lung adenocarcinoma. However, disease recurrence occurs in a third of patients. Prognostic biomarkers are needed to improve postoperative management. Here, we evaluate the utility of Homeobox A9 (HOXA9) promoter methylation, alone or in combination with Blood Vessel Invasion (BVI) assessment, for prognostic stratification of stage I lung adenocarcinoma patients.Materials and methodsWe developed a Droplet Digital PCR (ddPCR) assay to measure HOXA9 promoter methylation in formalin-fixed paraffin-embedded (FFPE) biospecimens generated during routine pathology. The prognostic value of HOXA9 promoter methylation and BVI, alone and in combination, was evaluated by Kaplan-Meier survival and Cox regression analyses in a cohort of 177 stage I lung adenocarcinoma patients from the NCI-MD study.ResultsThe ddPCR assay showed linearity, sensitivity and specificity for measuring HOXA9 promoter methylation down to 0.1% methylated DNA input. The HOXA9 promoter was methylated de novo in FFPE tumors (P < 0.0001). High methylation was independently associated with worse cancer-specific survival (Hazard Ratio [HR], 3.37; P = 0.0002) and identified high-risk stage IA and IB patients. Addition of this molecular marker improved a risk model comprised of clinical and pathologic parameters (age, gender, race, stage, and smoking history; nested likelihood ratio test; P = 0.0004) and increased the C-index from 0.60 (95% CI 0.51-0.69) to 0.68 (0.60-0.76). High methylation tumors displayed high frequency of TP53 mutations and other molecular characteristics associated with aggressiveness. BVI was independently associated with poor outcome (HR, 2.62; P = 0.054). A score that combined BVI with HOXA9 promoter methylation further stratified high-risk patients (trend P = 0.0001 comparing 0, 1 or 2 positive markers).ConclusionsddPCR can be used to quantify HOXA9 promoter methylation in FFPE samples. Alone or combined with BVI in a prognostic classifier, HOXA9 promoter methylation could potentially inform the clinical management of patients with early-stage lung adenocarcinoma.

Dataset Information

Performance Evaluation of SHOX2 and RASSF1A Methylation for the Aid in Diagnosis of Lung Cancer Based on the Analysis of FFPE Specimen.

Publications

Performance Evaluation of <i>SHOX2</i> and <i>RASSF1A</i> Methylation for the Aid in Diagnosis of Lung Cancer Based on the Analysis of FFPE Specimen.

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